Abstract

Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, we developed an assay to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, we quantified the frequency of telomere formation in different genetic backgrounds with known or possible defects in DNA damage repair. We showed that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. Our study identifies several pathways that suppress telomere addition at DSBs, paving the way for future mechanistic studies.

The terminal deficiency (TD) assay. (A) Genomic structure of the starting D4A chromosome is shown at the top, which has the wild-type Sco + gene and the dominant KrIF mutation. The structures of the D4A region in different progeny are shown below. The box with a long arrow depicts the mini-white marker gene, which is color coded in accordance with the different eye colors and patterns that it produces in different progeny. Schematic representation of the different eye colors and shapes are given in the “Eye” column. Different progeny classes are also defined by their phenotypes, which are given to the left of the eye diagrams. In the w + Kr + class, placement of white at the telomere resulted in a mottled pattern of eye pigmentation. In the w Kr + class, the white gene is 5′ truncated losing its expression. In the w + + Kr + class, a transposon (green block arrow) attached 5′ to white leading to its higher expression. In the w KrIF class, the deletion (parentheses) was generated during NHEJ repair, abolishing white expression. The positions of the cut site for EcoRV (R5) are given along with the location of the probe and the predicted size of the fragment, except in the case of w + + Kr + as the R5 location on the transposon is not known (question mark). The position of the PCR fragment used to test the presence of D4A sequence is shown for the w Kr + class of progeny. (B) Eye pictures of some of the progeny recovered with phenotypes given to the left.

Molecular analyses of events from the TD assay. (A) Southern blot analyses of terminal deletions from various genetic backgrounds. Genomic DNA was digested with EcoRV. “M” represents marker lanes with marker sizes in kilobases. Genotypes were shown at the top for the first three lanes. Lines 1–6 are from events with transposon attachment (w + + Kr + class in ) with the germline genotype as followed: 1, wt; 2, mu2; 3, tefu; 4, mus304; 5, Irbp; and 6, spnA. Bands from the endogenous white gene are denoted with asterisks. Note that both the w1118 and yw1 backgrounds were used in the experiments. Bands from mini white at D4A are labeled with arrowheads. (B) Southern blot analyses on TD flies of different generations from either wild-type (wt) or Irbp backgrounds. Bands from endogenous white are labeled with asterisks. The D4A band shows a steady decrease in sizes in both backgrounds. The extra white bands in the Irbp background originated from the w1 allele. (C) Design of inverse PCR to clone the very end of D4ATD. The small arrows depict the positions of the PCR primers. The hexagon represents the end of the chromosome. An EcoRV digestion followed by blunt-end ligation created a circular piece of DNA allowing productive PCR reactions. Partial sequences from three clones are given with bold-typed “GAT” separating the internal sequences from the terminal sequences (in italics). GAT represents half of the GATATC cut site for R5. The extent of end loss was estimated for these three lines starting from the position at which I-SceI cut. (D) PCR results from assaying the presence of D4A sequences in different progeny with phenetypes given at the top. Note that for one of the w Kr + events, the control PCR was positive but not for the D4A PCR. (E) Polytene telomeres from progeny recovered from the tefu germline in which the distal KrIF piece of DNA transposed to the tip of chromosome 3L. The flies were heterozygous for the transposition so that an extra bit of chromosomal material (arrow) is present on only one of the two homologous telomeres.

Frequency plots from the TD assay. The numerical data and statistical analyses were given in . The genotypes are given at the top of the charts. For experiments with a maternal contribution of I-SceI, both male (M) and female (F) parents were used. The top chart depicts total TD frequencies in different genetic backgrounds. *P value <0.0001 when compared to the wild-type sample. The middle chart depicts the frequencies of the appearance of TD progeny that had loss part of the white gene due to end degradation. **P value ≤0.001. The bottom chart depicts the level of transposonattachment to DSBs in different backgrounds. ***P value ≤0.05.